Separase (Esp1 in budding yeast) is a conserved protease that is necessary for chromosome segregation. Mice lacking separase function fail to survive and cell lines in which separase is inactivated show gross chromosome mis-segregation. During mitosis and meiosis, separase cleaves one of the subunits of the cohesin complex that links the two sister chromatids together. This cleavage leads to the dissolution of cohesion, allowing the sister chromatids to be pulled apart by the spindle microtubules. Separase is regulated at multiple levels, including phosphorylation, auto-cleavage, and sub-cellular localization. In addition, the activity of separase is regulated by an inhibitor, called securin (Pds1 in budding yeast), which binds to separase and blocks its active site. Evidence from both yeast and higher eukaryotes suggests that securin/Pds1 is not only an inhibitor but that its binding to separase is needed for separase activation. The mechanism of separase activation by Pds1, or any other protein, is unknown. In this project, we aim to uncover the mechanisms for separase activation, and in particular we are focusing on proteins and pathways that control separase's nuclear localization. Previous work from our labs and others showed that Pds1 is needed for Esp1s nuclear localization, but it is not known if Esp1 enters the nucleus unaccompanied by Pds1 and then gets sequestered in the nucleus through Pds1 binding, or whether Pds1 shuttles in and out of the nucleus, interacting with Esp1 in the cytoplasm and promoting its nuclear localization. There is also evidence to suggest that other proteins are involved in Esp1's nuclear localization: in mutants lacking Pds1, Esp1 enters the nucleus on schedule, albeit at reduced levels, and in wild type cells Esp1 lingers in the nucleus after Pds1 is removed by protein degradation. [unreadable] [unreadable] To gain a better understanding of how separase is activated and to uncover proteins involved in the nuclear localization of the budding yeast Esp1, we have taken a two pronged approach. The first, which is being done in collaboration with Dr. Mark Winey, is a genetic based approach making use of several different esp1 mutant alleles. The mutations in these alleles all fall in different ESP1 regions and result in different mutant phenotypes in terms of cell cycle progression and conditions for inactivation. It should be noted that apart from the protease domain, the functions of most of the Esp1 protein domains are unknown. We are currently undertaking a high copy suppressor screen, the idea being that by over expressing proteins that normally interact with Esp1 or lead to its activation, we will be able to overcome the defects of the various mutant alleles. Indeed, so far we have isolated multiple suppressors; some of which are common to all esp1 alleles and some are allele specific. Some suppressors act as protein chaperons, some increase the levels of Esp1 and some suppress by a yet unknown mechanism. Using this approach we will identify proteins that contribute to Esp1 activation and further our knowledge of separase function in higher eukaryotes. To study the regulation of Esp1s nuclear localization we are currently developing tools to detect Esp1 in live and fixed cells. We will then use these tools to determine the protein regions of Esp1 that are necessary for its nuclear localization, either in the presence or absence of Pds1. We will then identify proteins that facilitate the nuclear localization of Esp1 and determine the precise role of Pds1 in this process.